Although sickle cell disease and its clinical manifestations has been recognized within West Africa for several centuries, the first report of sickle cell anemia appearing in the medical literature occurred only in 1910 when James B. Herrick documented the presence of anemia in a 20-year-old black male using photomicrographs illustrating the presence of "thin sickle-shaped and crescent-shaped" red cells [Arch. Intern. Med. 6:517 (1910)]. Other cases of sickle cell disease were then continually recognized and reported over the next forty years until when in 1949 it was unequivocally confirmed that patients with sickle cell anemia had an electrophoretically abnormal hemoglobin, whereas those with the "sickle trait" had equal amounts of the normal and abnormal hemoglobin components. [Pauling, et al., Science 110:543-548 (1949)]. The inheritance pattern of other hemoglobin variants was subsequently clarified and provided convincing evidence that hemoglobin (Hb) S and hemoglobin (Hb) C are allelic variants of normal hemoglobin.
Sickle cell anemia and the existence of sickle hemoglobin (Hb S) was the first genetic disease to be understood at the molecular level; and is recognized today as the morphological and clinical result of the glycine to valine substitution at the No. 6 position of the beta globin chain [Ingram, V. M., Nature 178:792-794 (1956)]. The origin of the amino acid change and of the disease state is the consequence of a single nucleotide substitution [Marotta et al., J. Biol. Chem. 252:5040-5053 (1977)].
As sickle cell disease became better known and more easily identified, a remarkable degree of clinical heterogeneity in the physical manifestations and symptoms of sickle cell disease has become recognized. The anemia typically is of moderate severity and is usually well compensated by the dynamic steady state systems. The major source of mobidity and mortality is vaso-occlusion--which causes repeated episodes of pain in both acute and chronic form and also causes ongoing organ damage with the passage of time. Vascular occlusion often results in infarction of bone and/or bone marrow. Pulmonary and renal damage are frequently lethal in young adults; and cerebral infarction is often debilitating or fatal in children. Typically, patients afflicted with sickle cell disease are also very susceptible to bacterial infections and splenic dysfunction. Publications which describe the clinical and pathological manifestations in detail and review sickle cell disease are represented by the following: Clinton H. Joiner, Cation Transport And Volume Regulation In Sickle Red Blood Cells, American Journal of Physiology, 1992; Bunn, H. F. and B. G. FoRget, Hemoglobin: Molecular, Genetic and Clinical Aspects, U. B. Saunders Co., Philadelphia, 1986, Chapters 11 and 12, pages 453-564; Eaton, W. A. and J. Hofrichter, Blood 70:1245-1266 (1987); and Hebbel, R. P., Blood 77:214-237 (1991); and the reference cited within each of these publications.
It has long been recognized and accepted that the deformation and distortion of sickle cell erythrocytes upon complete deoxygenation is caused by polymerization and gelation of hemoglobin S. The phenomenon is well reviewed and discussed by Eaton and Hofrichter, Blood 70:1245 (1987). To gain some perspective on the problem and consequences of Hb S polymerization and intracellular gelation, it is useful to consider the events believed to occur as a red cell travels through the circulation of a patient afflicted with sickle cell disease. Erythrocytes containing no polymerized hemoglobin S in the arterial circulation may pass through the microcirculation and return to the lungs without sickling; or they may sickle in the veins; or they may sickle in the capillaries. For purposes of description, sickling is equivalent with intracellular gelation. The probability for each of these possible events for the sickle red cell will be determined by the delay time for intracellular gelation relative to the appropriate capillary transit time [Eaton et al., Blood 47:621 (1976)]. Thus, if it is thermodynamically impossible for intracellular gelation to take place, or if the delay time at venous oxygen pressures is longer than about 15 seconds, then cell sickling will not occur. Alternatively, if the delay time is between about 1 and 15 seconds, then the red cell will likely sickle in the veins. However, if the delay time is less than about 1 second, the red cell will sickle within the capillaries.
Note that for red cells that sickle within the capillaries, a number of possibilities exist as the consequent events--ranging from no effect on its transit time, to transient occlusion of the capillary, or to a more permanent blockage that may ultimately result in ischemia, or the infarction of the surrounding cells, and in destruction of the red cell. Which of these various possibilities and differing events will actually occur will depend on a number of factors: the total intracellular hemoglobin concentration; the composition of the intracellular hemoglobin; the rate and extent of deoxygenation; and the various transit times involved for the cells.
In addition, for unsickled red cells entering the microcirculation, a long capillary transit time will increase the probability of the potentially damaging vaso-occlusive events in two different ways. First, it will permit increased oxygen extraction which, in turn, will shorten the delay time. Second, it will increase the probability that a red cell with a given delay time will sickle within the capillary. Thus, for cells that either enter the microcirculation already sickled or become sickled within the microcirculation, there is a clear probability for occlusion of the small vessels; and the duration of an occlusion may be sufficiently long to compromise the oxygen supply to the surrounding tissues and hence alter the sickling and consequent vaso-occlusion in nearby microvessels. It is therefore critically important to recognize that vaso-occlusion is a dynamic process in which the fraction of capillaries that are occluded depends upon both rates of occlusion and the rate of capillary reopening. The factors that influence the transit times and the duration of occlusions thus play a critical role in the pathology in the sickle cell disease state.
It will also be noted and appreciated that the physical manifestations of sickle cell disease are paralleled by a cellular pathophysiology which is markedly diverse and varied. Certainly, much of the physiological dysfunction in sickle erythrocytes arises from the tendency of deoxy hemoglobin S to form an intracellular polymer--which results in a marked increase in cellular viscosity and impairment of rheological function. Sickle cells exhibit oxidative damage; abnormal adherence to endothelial cells, monocytes and other red cells; increased membrane rigidity; abnormal cytoskeleton function; deranged lipid structure; cation depletion and cellular dehydration; and abnormal carrier-mediated and passive permeability to cations.
Knowledge of the pathophysiology of sickle cells is merely one aspect of the continuing research interest in the physiology of erythrocyte generally. Considerable investigative efforts have focused upon the mechanisms of action and the various systems responsible for cation transport and volume regulation in normal red blood cells. In particular, the potassium transport pathways and the consequences of erythrocyte dehydration have been of major interest. A current summary of the various potassium transport pathways present in normal human erythrocytes is given by Table A below.
TABLE A __________________________________________________________________________ Potassium Transport Pathways in Human Erythrocytes* Maximal Capacity as K+ transporter System Mode (mmol/l.cells/h) Inhibitor Comments Reference __________________________________________________________________________ NA+/K+ pump Normally 3NA + 2K+ 1-3 Cardiac glycosides ATP-driven: Glynn, L. M., The but partial fluxes (ouabain) operates at approx. Enzymes of occur 50% V.sub.max at Biological cell [Na+] Membranes, 1985. NaKCI contransport 1Na+:1K+:2Cl- 0.1-1.5 Loop diuretics Poised at close Chipperfield, A. R., complex partial and (bumetanide, equilibrium (i.e., Clin. Sci. 71:465 exchange fluxes furosemide) zero net fluxes (1986). under physiological conditions) KCI cotransport 1K+:1Cl- &gt;10 Inernal divalent Highest in young Ellory, et al., cations; loop cells; activated Biomed. Biochem. diuretics at high NEM, pressure, Acta 46:531 (1987). concentrations swelling, acid pH. Ca.sup.2 +-activated K+ Uncoupled K+ &gt;10 Quinine Activated by Lew & Ferreira channel (Gardos cell [Ca.sup.2 +] Cur. Top. Memb. channel) Transp. 10:217 (1978). __________________________________________________________________________ *Source: Stuart, J. and J. C. Ellory, Brit. J. Hematol. 69:1-4 (1988).
It has long been recognized that the cytoplasm of the normal erythrocyte comprises approximately 70% water. Water crosses a normal erythrocyte membrane in milliseconds; however, the loss of cell water causes an exponential increase in cytoplasmic viscosity as the mean cell hemoglobin concentration (MCHC) rises above about 32 g/dl. Since cytoplasmic viscosity is a major determinate of erythrocyte deformability, the dehydration of the erythrocyte has substantial rheological consequences. Thus, the physiological mechanisms that maintain the water content of a normal erythrocytes, and the pathological conditions that cause loss of water from erythrocytes in the blood circulation, are critically important. Moreover, since cell water will follow osmotically any change in the intracellular concentration of ions, the maintenance of the red cell's potassium concentration is of particular importance [Stuart, J. and J. C. Ellory, Brit. J. Haematol. 69:1-4 (1988)].
Thus, the potassium transport pathways and the consequences of erythrocyte dehydration affect a number of different cell attributes: the ageing of normal erythrocytes [Brugnara, C. and D. C. Tosteson, Am. J. Physiol. 252:C269-C276 (1987)]; the quality of erythrocytes while stored in anticoagulant preservative solutions in the Blood Bank [Wallas, C. H., Transfusion 19:210-215 (1979)]; dehydration of both normal and abnormal red cells [Clark et al., Blood 51:1169-1178 (1978); Bookchin et al., J. Clin. lnvest., 87:113-124 (1991): and Lew et al., J. Clin. Invest., 87:100-112 (1991)]. Not surprisingly, as the different cationic transport mechanisms and pathways in human erythrocytes became known and better understood in detail, a variety of attempts were made to alter or influence the transport pathways. Merely representative of the reported attempts and to use various inhibitors to modify and alter the potassium transport pathways in human erythrocytes are those publications referenced and those inhibitors identified within Table A above. Clearly, different kinds and chemical classes of inhibitors have been experimentally investigated; and a range of different potencies for the various inhibitors were revealed. In addition, different modes of inhibition for the potassium transport pathways using a variety of different chemical agents have been reported in the literature. Merely representative of the current research investigations and publications in this field are the following: Turner et al. Vox Sanguinis 52:182-185 (1987); Alvarez et al., J. Biol. Chem. 267:11789-11793 (1992)]; Wolff et al., J. Membr. Biol. 106:243-252 (1988); Brugnara et al., J. Gen. Physiol. 100:47a (1992); Ellory et al., FEBS 296:219-221 (1992).
As regards sickle cell disease, the various attempts and approaches to therapeutically treating dehydrated sickle cells (and thus decreasing polymerization of hemoglobin S by lowering the osmolality of plasma) deserves special mention. The reported attemps have included the following approaches: intravenous infusion of distilled water [Knochel, J. T., Arch. Int. Med. 123:160-165 (1969)]; intravenous infusion of hypotonic saline [Gye et al., Am. J. Med. Sci. 266:267-277 (1973)]; administration of the antidiuretic hormone vasopressin together with a high fluid intake and salt restriction [Rosa et al., M. Eng. J. Med. 303:1138-1143 (1980); Charache, S. and W. G. Walker, Blood 58:892-896 (1981)]; the use of monensin to increase the cation content of the sickle cell [Clark et al., J. Clin. Invest. 70:1074-1080 (1982); Fahim, M. and B. C. Pressman, Life Sciences 29:1959-1966 (1981)]; intravenous administration of cetiedil citrate [Benjamin et al., Blood 67:1442-1447 (1986); Berkowitz, L. R. and E. P. Orringer, Am. J. Hematol. 17:217-223 (1984); Stuart et al., J. Clin. Pathol. 40:1182-1186 (1987)]; and the use of oxpentifylline [Stuart et al., J. Clin. Pathol. 40:1182-1186 (1987)].
Despite these many reports and research investigations, all of them are fairly said to be complete failures in being proved to be highly toxic, impractical as either prophylactic or therapeutic treatment regimens, and/or producing side-effects which outweighed the benefits and value of using them. There remains, therefore, a longstanding and well recognized need for an effective method of treating sickle erythrocytes and sickle cell disease utilizing substances which are not cytotoxic in use concentrations, are effective, and avoid major side-effects and complications. The development of such a treatment methodology would be recognized by clinicians and research investigators alike as a major breakthrough and achievement in this technical field.